Ubiquitin C-terminal hydrolases (UCHs) are a family of cysteine hydrolases that catalyze the hydrolysis of amides, esters and thioesters of the C-terminus of ubiquitin. Mammalian neuronal cells abundantly express a deubiquitylating enzyme, ubiquitin carboxy-terminal hydrolase 1 (UCH-L1). Mutations in UCH-L1 are linked to Parkinson's disease as well as gracile axonal dystrophy (gad) in mice. In contrast to the universally expressed UCH-L3 isozyme, UCH-L1 is expressed exclusively in neurons and testis/ovary. It has been shown that UCH-L1 associates and co-localizes with monoubiquitin and elongates ubiquitin half-life and the suggestion is made that UCH-L1, with avidity and affinity for ubiquitin, ensures ubiquitin stability within neurons.

Add 90 µl of Assay Buffer to the desired number of microplate wells and warm to assay temperature (e.g. 25°C).

Add 5 µl of diluted UCH-L1 and incubate for 30 min. to allow time for DTT-mediated activation of the enzyme.

Start the reaction by adding 5 µl of the 20x ubiquitin-AMC and mixing well. The Km of this preparation of UCH-L1 for ubiquitin-AMC was determined to be 57 nM and assays are typically performed at final concentrations in the range of 0-1 µM.

Monitor the progress of the reaction by measuring the fluorescence of the AMC cleaved from the ubiquitin-AMC. For example, read fluorescence (Ex. 350-380 nm; Em. 440-460) at 1 min. intervals for 10 min.

Determine the fluorescence in wells filled with 100 µl of a series of dilutions of AMC in Assay Buffer (0-1 µM). Plot AMC concentration (µM, y-axis) as a function of fluorescence (arbitrary fluorescence units, AFU, x-axis).

Data analysis: A conversion factor relating fluorescence to AMC concentration may be determined from the slope of the line (µM/AFU) obtained in step 5). Initial rates of ubiquitin-AMC cleavage by UCH-L1 may be determined from the slope (AFU/min) of the early, linear part of the reaction progress curve. A rate in, for example, pmol/min, can then be calculated by multiplying the initial slope of the progress curve by the slope of the calibration curve and the reaction volume (100 µl): Rate (pmol/min) = (AFU/min) x (µM/AFU) x (100 µl)